Structure-Function Studies of the Receptor Tyrosine Kinase MuSK

受体酪氨酸激酶 MuSK 的结构-功能研究

• 批准号: 7164429
• 负责人: STEVAN R. HUBBARD
• 金额: $ 33.23万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7164429
• 关键词: Acetylcholine; Agrin; Binding; Biochemical; Biological Assay; Biological Process; Cell Nucleus; Cholinergic Receptors; Chromosome Pairing; Crystallography; Cysteine-Rich Domain; Cytoplasmic Tail; Defect; Development; Event; Genes; Goals; Helix (Snails); Heparan Sulfate Proteoglycan; Immunoglobulins; In Vitro; Integral Membrane Protein; Length; Molecular; Motor Neurons; Muscle Contraction; Muscle Fibers; Muscle Proteins; Muscle-Specific Kinase; Neuromuscular Diseases; Neuromuscular Junction; Play; Process; Receptor Protein-Tyrosine Kinases; Research Personnel; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Synapses; Tyrosine Kinase Domain; Up-Regulation; computerized data processing; ear helix; extracellular; human FZD3 protein; mutant; peripheral membrane protein 43K; postsynaptic; presynaptic; programs; receptor; src-Family Kinases; three dimensional structure

DESCRIPTION (provided by applicant): The neuromuscular junction (NMJ) is a highly specialized synapse whose formation is dependent on MuSK (muscle-specific kinase), a receptor tyrosine kinase that is activated by agrin, a heparan-sulfate proteoglycan derived from motor neurons. MuSK activation results in the redistribution of muscle proteins to the postsynaptic site, including the acetylcholine receptor (AChR), rapsyn, ErbBs and MuSK itself. In addition, agrin-induced MuSK activation leads to selective transcriptional upregulation of synapse-specific genes in subsynaptic nuclei, and to induction of a retrograde signal leading to presynaptic differentiation. In mature NMJs, acetylcholine released from motor neurons activates AChRs and triggers muscle contraction. Both the ectodomain and cytoplasmic domain of MuSK play essential roles in signaling processes leading to formation of the NMJ. However, the molecular/structural mechanisms underlying these signaling processes are not understood. The goals of this proposal are to elucidate the structural mechanisms governing: (i) MuSK activation by agrin; (ii) MuSK co-clustering with the AChR and rapsyn, and (iii) MuSK recruitment of the non-receptor tyrosine kinases Src and Abl. The specific aims of this proposal are: Aim 1. Structural and functional characterization of the MuSK ectodomain Aim 2. Molecular characterization of the modes of interaction between MuSK and the non-receptor tyrosine kinases Src and Abl To achieve these aims, we will employ x-ray crystallography to determine the three-dimensional structures of the MuSK ectodomain and cytoplasmic domain. We will then characterize the functional roles of key residues identified from the structural studies by expressing select MuSK mutants in MuSK-deficient myotubes and assaying for agrin-induced MuSK activation, AChR clustering, and recruitment of Src and Abl to MuSK. Relevance: Formation of the NMJ is a fundamental biological process which is critical for organismal development. By revealing the biochemical mechanisms underlying the formation of this important synapse, we will better understand, at a molecular level, how defects in this process give rise to neuromuscular disorders.

描述（由申请人提供）：神经肌肉接头（NMJ）是一种高度特化的突触，其形成依赖于MuSK（肌肉特异性激酶），MuSK是一种受体酪氨酸激酶，由聚集蛋白激活，聚集蛋白是一种源自运动神经元的硫酸乙酰肝素蛋白聚糖。MuSK激活导致肌肉蛋白重新分布到突触后位点，包括乙酰胆碱受体（AChR），rapsyn，ErbBs和MuSK本身。此外，聚集蛋白诱导的MuSK激活导致突触下核中突触特异性基因的选择性转录上调，并诱导导致突触前分化的逆行信号。在成熟的NMJ中，运动神经元释放的乙酰胆碱激活AChR并触发肌肉收缩。 MuSK的胞外域和胞质结构域在导致NMJ形成的信号传导过程中起重要作用。然而，这些信号传导过程的分子/结构机制尚不清楚。本提案的目标是阐明以下结构机制：（i）聚集蛋白激活MuSK;（ii）MuSK与AChR和rapsyn共聚簇，以及（iii）MuSK募集非受体酪氨酸激酶Src和Src。本提案的具体目标是： 目标1. MuSK胞外域的结构和功能表征 目标2. MuSK与非受体酪氨酸激酶Src和Abl相互作用模式的分子表征 为了实现这些目标，我们将采用X射线晶体学来确定MuSK胞外域和胞质结构域的三维结构。然后，我们将通过在MuSK缺陷型肌管中表达选择MuSK突变体并测定聚集蛋白诱导的MuSK激活、AChR聚集以及Src和Abl向MuSK的募集来表征从结构研究中鉴定的关键残基的功能作用。 相关性：NMJ的形成是一个基本的生物过程，对生物体发育至关重要。通过揭示这一重要突触形成的生化机制，我们将在分子水平上更好地理解这一过程中的缺陷如何导致神经肌肉疾病。